Hexavalent chromium [Cr(VI)] is a potent human carcinogen found in the workplace of more than 0.5 million individuals in the US. Environmental exposure to Cr(VI) also represents a significant public health concern due to the presence of this metal in drinking water for millions of US households and at many Superfund toxic sites. Intracellular reduction of Cr(VI) results in the formation of a wide assortment of DNA lesions, including several forms of Cr-DNA adducts. We have previously found that Cr-DNA binding was responsible for all mutagenic and genotoxic effects generated by direct metabolism of Cr(VI) with its main biological reducers. The genotoxicity of Cr-DNA adducts was unexpected considering their very weak duplex-distorting properties reflecting attachment of Cr atom to the DNA phosphate group. Cr(VI) also causes very toxic, cancer-promoting DNA double-strand breaks (DSB) that is detectable at environmentally relevant Cr(VI) doses and poorly repaired. This application, which is built on our previously published findings and new preliminary data, is designed to investigate a mechanism of DSB formation via toxic DNA repair of Cr-DNA adducts. The proposed studies aim to identify specific forms of Cr-DNA damage that trigger toxic DNA repair and DSB formation, characterize nuclease activities involved in DNA strand cleavage and determine genomic host-spots for DNA breakage and DNA repair processes. A successful completion of this work can help identify toxicologically important biomarkers of Cr(VI) exposure and high-risk genomic sites that can be used for monitoring of genetic damage in exposed populations.